There are two major parts to this proposal: 1. To define the overall superoxide-and peroxide-producing oxidase system of human and guinea pig granulocytes, and to elucidate the mechanism that stimulates the oxidase in the plasmalemma. 2. To determine the biochemical concomitants of activation of mouse macrophages, when these are exposed to separated fractions of culture fluids of sensitized lymphocytes. Experiments will also be performed on biochemical changes in macrophages after the two cell-types have been in direct contact. The objectives are to comprehend, in granulocytes, the metabolic basic of the major microbicidal system, and in the macrophages, the biochemical basis of activation ( i.e., enhanced efficiency) and of the stimulated secretion of a mitogenic factor. In the first series of experiments we shall extract the plasmalemmal oxidase with detergent, purity by exclusion chromatography and gel-electrophoresis, and raise antibodies against this enzyme and/or soluble cytoplasmic protein that also produces 02 ion and H202. These will assist in purification and in assessing physiological importance. We shall use F minus as stimulating agent in double-labeling experiments to determine whether components of the membrane are phosphorylated (or dephosphorylated) when respiration is increased. In the second series of studies we shall obtain MIF-rich filtrates, fractionate these on G-100 columns, and by other methods. Fractions will be added to cultured peritoneal macrophages, and antibacterial, tumoricidal, oxidative (glucose) and ecto-AMPase activities determined as well as cell migration. The fractionation and identification of filtrate components will be facilitated by culturing the sensitized or control lymphocytes in media whose components are labeled with 14C or 3H respectively, and following ratios of isotopes in products.